The general process of incorporating or injecting biomaterials into animal and plant cells or other microorganisms is commonly referred to as transfection. There are several known modes or methods of transfection, often referred to as “delivery” or “biodelivery” methods. Laser optoinjection (also called optoporation or photoporation) is a laser-mediated biodelivery method of transfection wherein biomolecules and other biomaterials (e.g., recombinant DNA, RNA, proteins, and nanoparticles) are incorporated into microbiological organisms (referred to as biological targets) through the outer bi-lipid protecting membrane of cells or other biological targets. Optoinjection can also include the injection of biomolecules through the nuclear membrane of animal and plant cells and eukaryotic microorganisms.
In laser optoinjection transfection, laser light is focused onto a protective barrier (such as a bi-lipid membrane) of a biological target, which causes a localized increase in the barrier's permeability. The localized increase in permeability at the focal point of the laser light allows for the diffusion (or other form of mass transport) of materials into the biological target. Such techniques are desirable to injecting materials into the biological target where the materials cannot otherwise penetrate the protecting barrier of the target. The increase in permeability is only temporary and, in most cases, decreases back to normal in a relatively short span of time. Laser optoinjection is classified as a physical delivery method since transfection (injection) is mediated through the direct structural disruption in a protecting barrier by the laser.
To achieve transfection using laser optoinjection, the laser light must be focused onto the membrane of the biological target with an oil-immersion objective lens that has a high numerical aperture (and thereby a short focal length). Due to the high numerical aperture and associated very short focal length of an oil-immersion lens, the biological target to be irradiated and injected with the laser must be stably positioned within approximately 200 micrometers (±100 micrometers) of the objective. The stable positioning constraints involved in laser optoinjection presents a huge challenge in transfection of biological targets, and is, thus, a major reason why a device for stabilization of the target is necessary for successful commercialization and use of laser optoinjection. The prior art, however, does not disclose adequate techniques or devices for the required stabilization of biological targets.
The inability to transfect biological entities that do not stick to culture surfaces via laser-mediated biodelivery (optoinjection) with limited interference is a major hindrance of the technique(s). Biological entities/targets (cells or other microorganisms) that do not adhere to surfaces are non-adherent. A vast array of non-adherent entities have potential as transfection targets in the modern biotechnology industry/space. The laser optoinjection technique also suffers from extremely low throughput since no robust platform exists to automate the transfection process. Automation would increase injection rates and enable laser-mediated transfection at a large scale. Other significant challenges facing current laser optoinjection methods include difficulties in re-focusing the laser on each biological target, which requires high throughput or the injection of targets that are not closely spaced (i.e., tightly packed together) thereby causing high injection efficiencies (% of cells transfected relative to the number irradiated by the laser). At present, no platform or method exists to enable the laser optoinjection transfection to overcome the above limitations and maintaining compatiblility with desirable enhancements to laser-mediated transfection including in-situ culture, post-injection monitoring/imaging, and intracellular optical tweezing (e.g., to enhance nuclear transfection in eukaryotic entities).